WO2024034559A1 - Method for producing cell aggregates - Google Patents

Abstract

The present invention provides a method for mass producing cell aggregates such as artificial organoids through an approach that differs entirely from previous methods. Specifically, provided is a method for producing cell aggregates that includes a step in which a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells are filled into microfibers, and the mixture is suspension cultured to form cell aggregates.

Description

Method for producing cell aggregates

The present invention relates to a method for producing cell aggregates. More specifically, the present invention relates to a method for producing a cell aggregate, which includes a step of filling a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells into microfibers and culturing them in suspension to form a cell aggregate.

For serious organ failure, organ transplantation and artificial organ replacement treatments are performed in clinical practice. However, fundamental unresolved issues remain, such as organ transplants being subject to rejection and an absolute shortage of donors, and artificial organs only being able to replace part of their functions for a short period of time. Therefore, methods are being developed to induce artificial organoids that can withstand transplantation. For example, in Patent Document 1, by artificially reproducing the early process of organ development, early differentiation is induced through interactions between multiple cell lineages, and tissue forming ability of organ cells that have undergone early differentiation is induced. However, they have succeeded in artificially creating organ buds, which are the source of tissues and organs, in vitro. Specifically, in Patent Document 1, a cell suspension containing liver endoderm cells or pancreatic β cell lines, vascular endothelial cells, and mesenchymal stem cells is placed in a culture dish immobilized with Matrigel. By sowing upward, organ buds are produced. However, the method of Patent Document 1 is not suitable for mass production of organ buds.

In addition, new culture methods have been developed that imitate the surrounding environment and morphology of cells in vivo to obtain cells with functions closer to those in vivo. As such a culture method, the spheroid culture method, which is a method that can maintain cell interaction, has attracted attention, and has been applied to the culture of various cells such as pancreatic islet cells, liver cells, stem cells, and cancer cells. For culturing spheroids, there are two methods: forming spheroids individually by leaving cell populations in round-bottomed wells (hanging drop method; e.g., Non-Patent Document 1), and forming droplets in wells with openings. Among them, there are methods for forming spheroids (for example, Patent Document 2), but these methods have advantages and disadvantages, such as being unsuitable for mass production of spheroids and being limited by the size and type of cells of the spheroids.

International Publication No. 2013/047639 International Publication No. 2015/129263

Sasitorn Rungarunlert et al., “Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors”, World Journal of Stem Cells 1(1), December 31, 2009, pp.11-21

Therefore, an object of the present invention is to provide a method for mass-producing cell aggregates such as artificial organoids using an approach completely different from conventional methods.

The present inventors attempted to develop a method for efficiently producing cell aggregates such as organoids containing multiple types of cells by improving the conventional suspension culture for producing spheroids. However, in conventional spheroid culture methods, when a cell population containing multiple types of cells is cultured in suspension, the same types of cells aggregate with each other, making it impossible to obtain the desired cell aggregates containing multiple types of cells. (For example, Junji Fukuda Research Report, 2019, Takechi et al, DEVELOPMENTAL BIOLOGY 37,340-350 (1981)) We came up with the idea that by filling a mixture of multiple types of cells and culturing them in suspension, we could prevent the aggregation of cells of the same type and obtain cell aggregates containing multiple types of cells.Based on this idea, we , when a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells was filled in microfibers and cultured in suspension, they succeeded in obtaining cell aggregates containing each cell evenly. We found that the cell aggregates are structures that can be called organoids that have new functions (e.g., albumin secretion ability) that the original cells do not have.Based on these findings, the present inventors As a result of further research, we have completed the present invention.

That is, the present invention is as follows.
[1]
A method for producing a cell aggregate, comprising the steps of filling a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells into microfibers and culturing them in suspension within the microfibers to form cell aggregates.
[2]
The method according to [1], wherein the cell aggregate is an organoid.
[3]
The method according to [1] or [2], wherein the organ cell is an endodermal cell.
[4]
The method according to any one of [1] to [3], wherein the endodermal cells are hepatocytes.
[5]
The method according to any one of [1] to [4], wherein the microfiber has an inner diameter of 20 to 300 μm.
[6]
The method according to any one of [1] to [5], wherein the cell density within the microfiber during cell filling is 5 x 10 ⁶ cells/mL to 4 x 10 ⁸ cells/mL.
[7]
The method according to any one of [1] to [6], wherein the proportion of mesenchymal stem cells in the cell population at the time of cell filling is 1% or more and 20% or less.
[8]
The method according to any one of [1] to [7], wherein the proportion of vascular endothelial cells in the cell population at the time of cell filling is 10% or more and 60% or less.
[9]
The method according to any one of [1] to [8], wherein at least one of mesenchymal stem cells, vascular endothelial cells, and organ cells is derived from induced pluripotent stem cells.
[10]
A cell aggregate produced by the method according to any one of [1] to [9].
[11]
A transplant therapeutic agent comprising the cell aggregate according to [10].
[12]
The transplant therapeutic agent according to [11] for treating tissue or organ damage or disease.
[13]
A method for treating tissue or organ damage or disease, which comprises administering or transplanting an effective amount of the cell aggregate according to [10] into a subject.
[14]
The cell aggregate according to [10] for use in treating tissue or organ damage or disease.
[15]
Use of the cell aggregate according to [10] in the production of a therapeutic agent for tissue damage or disease.

According to the present invention, cell aggregates containing multiple types of cells can be produced in large quantities. Furthermore, since the cell aggregates produced according to the present invention can function as organoids, they can be useful as therapeutic agents for transplantation into living bodies.

FIG. 2 is a diagram showing the results of observing spheroids produced using Elplasia plates using a fluorescence microscope. It is a figure which shows the result of observing the spheroid manufactured with the hollow microfiber with the fluorescence microscope. FIG. 3 is a diagram comparing the amount of albumin secreted per unit cell number secreted by each spheroid.

1. TECHNICAL FIELD The present invention relates to a method for producing cell aggregates , which includes a step of culturing a plurality of types of cells under suspension culture conditions. More specifically, a method for producing cell aggregates (hereinafter referred to as (sometimes referred to as "the manufacturing method of the present invention"). In another embodiment, a cell aggregate obtained by the production method of the present invention is also provided.

As used herein, "microfiber" refers to a structure that includes a core portion and a sheath-like shell (coating) portion that covers the core portion, and is typically a hollow microfiber. In the production method of the present invention, mesenchymal stem cells, vascular endothelial cells, and organ cells are cultured in suspension in the hollow portion (ie, core portion) of the microfiber. Therefore, in addition to various cells, the core portion contains a culture medium as described below. Further, the sheath-like shell portion may have a single layer structure containing a substance as described below, or may have a multilayer structure of two or more layers.

The material used for the sheath-like shell part is not particularly limited as long as it has the strength to hold the core part, and examples thereof include alginate, agarose, and the like. More specifically, examples include sodium alginate, potassium alginate, ammonium alginate, and combinations thereof. Further, for example, it may be a mixture of alginate and agar, agarose, polyethylene glycol, polylactic acid, nanocellulose, or the like. Alginic acid may be a natural extract or may be chemically modified. Examples of chemically modified alginic acid include methacrylate-modified alginic acid.

Further, as the substance used for the sheath-like shell part, there may be mentioned a substance that gels when it comes into contact with the above-mentioned alginate, agarose, etc. Specific examples include polyvalent cationic substances, and more specific examples include calcium chloride, barium chloride, and the like.

The inner diameter of the microfiber used in the production method of the present invention, that is, the inner diameter of the shell portion, can be appropriately set based on the size of the target cell aggregate, and is, for example, 10 μm to 500 μm, preferably 20 μm to 300 μm. That's about it. Further, the cross-sectional shape of the microfiber is preferably circular, but may be oval or polygonal such as a quadrangle or a pentagon. The length of the microfiber can be appropriately set based on the size of the target cell aggregate, and is, for example, about 1 mm to 100 m. Furthermore, the outer diameter of the microfiber, ie, the outer diameter of the shell portion, can be easily set appropriately based on the size of the target cell aggregate, and is, for example, about 10 μm to 2000 μm.

It is preferable to use sodium alginate and calcium chloride as the materials used for the shell portion of such a microfiber, and the shell portion preferably has a circular cross-sectional shape with an inner diameter of about 50 to 200 μm.

Such a microfiber can be produced by a known method (eg, JP 2017-99303, JP 2021-016319, etc.). In addition, although we do not wish to be bound by any theory, the reason why culturing in microfibers prevented the aggregation of cells of the same type and resulted in cell aggregates containing multiple types of cells is that It is presumed that this is because mixed cells in the vicinity of the microfiber can exist without spreading, so even cells of different types with different adhesion strengths were able to aggregate. Therefore, even microfibers other than those used in this example can be used in the present invention.

As used herein, "suspension culture conditions" refer to conditions under which cells or cell aggregates remain suspended in the culture medium, i.e., conditions under which cells or cell aggregates and microfiber core parts are maintained. Refers to conditions that do not allow direct or indirect strong cell-substratum junctions to be formed with the inner wall.

In addition, in the production method of the present invention, the proportion of organ cells in the cell population at the time of cell filling can be appropriately set based on the size and properties of the desired cell aggregate, but for example, it is 30% or more and 90% or less. Yes, preferably 45% or more and 77% or less.

A medium under suspension culture conditions can be prepared by adding medium additives to a basal medium as necessary. Examples of the above basal medium include RPMI-1640 medium, Eagle's MEM (EMEM), Dulbecco's modified MEM (DMEM), Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, Hybridoma Serum free medium, KnockOut ^TM DMEM (KO DMEM), Advanced ^TM medium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM/F-12), Chemically Defined Hybridoma Serum Free medium, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM/ F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g. : Waymouth's MB752/1), CMRL medium (e.g. CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCell Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC) ), ReproMed ^TM iPSC Medium (Reprocell Co., Ltd.), Cellartis MSC Xeno-Free Culture Medium (Takara Bio Co., Ltd.), TESR-E8 (Veritas Co., Ltd.), StemFit (registered trademark) AK02N, AK03N (Ajinomoto Co., Ltd.) and mixtures thereof, but are not limited to these.

Additionally, physiologically active substances and nutritional factors necessary for cell survival or proliferation can be added to the medium as necessary. These medium additives may be added to the medium in advance or during cell culture. The addition method during culturing may be any method such as adding one type of solution one by one or adding a mixed solution of two or more types, and may be continuous or intermittent addition.

Physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.) , various hormones, and various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.). Nutritional factors include sugars, amino acids, vitamins, hydrolysates, or lipids. Examples of the sugar include glucose, mannose, fructose, etc., and one type or a combination of two or more types may be used. Amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, and L-lysine. , L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, or L-valine, which may be used alone or in combination of two or more. Examples of vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, or DL-α-tocopherol, and one or more of them may be used in combination. It is used as Examples of hydrolysates include those obtained by hydrolyzing soybeans, wheat, rice, peas, corn, cottonseed, yeast extracts, and the like. Examples of lipids include cholesterol, linoleic acid, and linolenic acid. Examples of polysaccharides include gellan gum, deacylated gellan gum, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylamylose, xanthan gum, alginic acid, carrageenan, diutan gum, and locust bean gum.

Furthermore, an antibiotic such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the medium as necessary. When adding an acidic substance such as sialic acid to a medium, it is desirable to adjust the pH of the medium to a neutral range suitable for cell growth, pH 5 to 9, preferably pH 6 to 8.

The medium used for suspension culture may be a serum-containing medium (for example, fetal bovine serum (FBS), human serum, horse serum) or a serum-free medium. As the serum, FBS is preferred. From the viewpoint of preventing contamination with components derived from a different species of animal, it is preferable to use serum that does not contain serum or is derived from an animal of the same species as the cells to be cultured. Here, the serum-free medium means a medium that does not contain unadjusted or unpurified serum. The serum-free medium may contain purified blood-derived components or animal tissue-derived components (eg, growth factors).

Similarly to serum, the medium used for suspension culture may or may not contain a serum substitute. Serum substitutes include, for example, albumin, albumin substitutes such as lipid-rich albumin and recombinant albumin, vegetable starches, dextran, protein hydrolysates, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace amounts. 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Specific examples of serum substitutes include those prepared by the method described in WO98/30679, commercially available Knockout Serum Replacement [KSR] (Life Technologies), Chemically-defined Lipid concentrated (Life Technologies), and L -Alanine-L-glutamine dipeptide (e.g. Glutamax (Life Technologies)), etc. In addition, examples of biologically derived factors include platelet-rich plasma (PRP) and culture supernatant components of human mesenchymal stem cells.

From the viewpoint of efficient production of cell aggregates, the medium used for suspension culture preferably contains the above-mentioned polysaccharides, particularly methylcellulose. The concentration of methylcellulose in the medium is not particularly limited, but is, for example, 0.05% to 3% (w/v), preferably 0.3% (w/v).

As used herein, "organ cells" refer to functional cells that constitute an organ or undifferentiated cells that differentiate into functional cells. Examples of "undifferentiated organ cells" include cells that can differentiate into organs such as kidney, heart, lung, spleen, liver, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, and spinal cord. Cells that can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, crystalline lens, spleen, kidney, ureter, heart, blood, gonads, Cells that can differentiate into mesodermal organs such as adrenal cortex, muscle, skeleton, dermis, connective tissue, and mesothelium, liver, pancreas, gastrointestinal tract (pharynx, esophagus, stomach, intestinal tract), lungs, thyroid, parathyroid glands, urinary tract , cells that can differentiate into endodermal organs such as the thymus. Whether a cell is capable of differentiating into an ectodermal organ, mesodermal organ, or endodermal organ can be confirmed by examining the expression of marker proteins (if one or more of the marker proteins is expressed). If this is the case, it can be determined that the cells are capable of differentiating into endodermal organs.) For example, for cells that can differentiate into the liver, markers include HHEX, SOX2, HNF4A, AFP, ALB, etc., and for cells that can differentiate into the pancreas, markers include PDX1, SOX17, SOX9, etc., and cells that can differentiate into the intestinal tract. CDX2, SOX9, etc. are markers for cells that can differentiate into kidney, SIX2, SALL1, NKX2-5 for cells that can differentiate into heart, MYH6, ACTN2, MYL7, HPPA, and cells that can differentiate into blood. C-KIT, SCA1, TER119, HOXB4, and for cells that can differentiate into the brain and spinal cord, HNK1, AP2, and NESTIN are markers. Among the terms used among those skilled in the art: hepatoblast, hepatic progenitor cells, pancreatoblast, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm , metanephric mesenchymal precursor cells, multipotent nephron progenitor, renal progenitor cell, cardiac mesoderm, cardiovascular progenitor cells, cardiac progenitor cells, (JR. Spence, et al. Nature.;470(7332):105-9.(2011), Self , et al. EMBO J.; 25(21): 5214-5228.(2006), J. Zhang, et al. Circulation Research.; 104: e30-e41(2009), G. Lee, et al. Nature Biotechnology 25, 1468 - 1475 (2007)) and the like are included in the undifferentiated organ cells in the present invention. Undifferentiated organ cells can be produced from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. For example, organ cells that can differentiate into liver are K. Si-Taiyeb, et al. Hepatology, 51 (1): 297- 305(2010), T. Touboul, et al. Hepatology. 51(5):1754- 65. (2010), and organ cells capable of differentiating into pancreas can be produced according to D. Zhang, et al. Cell Res.;19(4):429-38.(2009). , organ cells capable of differentiating into the intestinal tract are J. Cai, et al. J Mol Cell Biol.;2(1):50-60(2010), R. Spence, et al. Nature.;470(7332): 105-9. (2011), organ cells capable of differentiating into the heart can be produced according to J. Zhang, et al. Circulation Research.; 104: e30-e41 (2009), and the brain Cells capable of differentiating into spinal cord or spinal cord can be produced according to G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007). "Differentiated organ cells" include endocrine cells of the pancreas, pancreatic ductal epithelial cells of the pancreas, hepatocytes of the liver, epithelial cells of the intestinal tract, tubular epithelial cells of the kidney, glomerular epithelial cells of the kidney, cardiomyocytes of the heart, and blood cells. Examples include lymphocytes and granulocytes, red blood cells, brain neurons and glial cells, spinal cord neurons, and Schwann cells. The organ cells mainly used are those derived from humans, but also from animals other than humans, such as rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, and ungulates such as pigs, cows, goats, and sheep. Cells of animals such as eyes, cats, dogs, cats, humans, monkeys, rhesus monkeys, marmosets, orangutans, primates such as chimpanzees, and the like may be used.

In the production method of the present invention, organ cells produced (differentiation-induced) from pluripotent stem cells may be used. For example, pluripotent stem cells are cultured in a medium containing Activin A to induce endoderm, and then cultured in a medium containing BMPs (BMP4, etc.) and FGFs (bFGF, FGF4, etc.) to induce hepatic progenitor cells. (HE) can be induced.

As used herein, "vascular endothelial cells" refer to cells that constitute vascular endothelium or cells that can differentiate into such cells. Whether a cell is a vascular endothelial cell or not can be confirmed by examining whether marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD31 are expressed (any of the above marker proteins If one or more of these are expressed, it can be determined that the cells are vascular endothelial cells). The vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether vascular endothelial cells are differentiated cells can be confirmed by CD31 and CD144. Among the terms used among those skilled in the art are endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, hemangioblast (HJ. joo, et al. Blood. 25;118(8):2094 -104.(2011)) and the like are included in the vascular endothelial cells in the present invention. The vascular endothelial cells used are mainly those derived from humans, but those derived from animals other than humans, such as rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, pigs, cows, goats, and sheep, are also used. Vascular endothelial cells derived from animals such as odorants, felids such as dogs and cats, humans, primates such as monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees may be used.

In the production method of the present invention, vascular endothelial cells produced (differentiation-induced) from pluripotent stem cells may be used. For example, pluripotent stem cells can be cultured in a medium containing BMP (such as BMP4) to induce mesoderm, and then cultured in a medium containing VEGF to induce vascular endothelial cells.

As used herein, "mesenchymal stem cells" refer to stem cells that have the ability to differentiate into cells belonging to the mesenchymal system. Whether a cell is a mesenchymal stem cell can be confirmed by examining whether marker proteins such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin are expressed ( If any one or more of the above marker proteins is expressed, it can be determined that the cells are mesenchymal stem cells). Among the terms used among those skilled in the art, mesenchymal stem cells, mesenchymal progenitor cells (R. Peters, et al. PLoS One. 30;5(12):e15689.(2010)), etc. refer to mesenchymal stem cells in the present invention. Included in lineage stem cells. Mesenchymal stem cells are mainly derived from humans, but may also be derived from animals other than humans, such as rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, pigs, cows, goats, and sheep. Mesenchymal stem cells derived from animals such as ungulates, cats such as dogs and cats, humans, primates such as monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees may be used.

In the production method of the present invention, mesenchymal stem cells produced (differentiation-induced) from pluripotent stem cells may be used. For example, pluripotent stem cells are cultured in a medium containing BMPs (such as BMP4), then cultured in a medium containing PDGF-BB and Activin A, and then cultured in a medium containing BMPs (such as BMP4). This makes it possible to induce mesenchymal stem cells.

"Pluripotent stem cells" are cells that can differentiate into tissues and cells with various different forms and functions in living organisms, and can be differentiated into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). It also refers to stem cells that have the ability to differentiate. Examples of the pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and embryos derived from cloned embryos obtained by nuclear transfer. Examples include nuclear transfer embryonic stem cells (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ cells (EG cells), but preferred are iPS cells (more preferably human iPS cells). If the above-mentioned pluripotent stem cells are ES cells or any cells derived from human embryos, the cells may be produced by destroying embryos, or they may be produced without destroying embryos. However, cells produced without destroying the embryo are preferable.

ES cells are stem cells that are established from the inner cell mass of early embryos (e.g. blastocysts) of mammals such as humans and mice and have pluripotency and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and ES cell lines were subsequently established in humans, monkeys, and other primates (J.A. Thomson et al. al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod ., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by removing an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage before the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113- 117), and can also be established using developmentally arrested embryos (Zhang X. et al. (2006), Stem Cells 24: 2669-2676.).

nt ES cells are cloned embryo-derived ES cells produced by nuclear transfer technology, and have almost the same characteristics as fertilized egg-derived ES cells (Wakayama T. et al. (2001), Science, 292 :740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). That is, nt ES (nuclear transfer ES) cells are ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To create nt ES cells, a combination of nuclear transfer technology (Cibelli J.B. et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (described above) is used (Wakayama et al. Kiyoka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (special issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated, unfertilized mammalian egg, and the egg can be initialized by culturing it for several hours.

As the ES cell line used in the present invention, various mouse ES cell lines established by inGenious targeting laboratory, RIKEN, etc. can be used, and human ES cell lines can be used. For example, various human ES cell lines established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used. Specifically, for example, human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, and H1 and H9 strains distributed by WiCell Research. Examples include the KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, and SSES3 strain distributed by RIKEN.

iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, and Yamanaka et al. established iPSCs (Takahashi K, Yamanaka et al. S., Cell, (2006) 126: 663-676), as well as human cell-derived iPSCs (Takahashi K, Yamanaka S., et al. Cell), which were established by introducing the same four factors into human fibroblasts. , (2007) 131: 861-872.) After introducing the above four factors, Nanog-iPSCs were selected and established using Nanog expression as an indicator (Okita, K., Ichisaka, T., and Yamanaka, S. (2007 ). Nature 448, 313-317.), c-Myc-free iPSCs (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), virus-free method iPSCs established by introducing 6 factors (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) etc. can also be used. In addition, induced pluripotent stem cells (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells created by Sakurada et al. (Unexamined Japanese Patent Publication No. 2008-307007), etc. can also be used.

In addition, all published papers (e.g. Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5,568-574;, Kim JB., Scholer HR., et al ., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, 795-797), or patents (for example, JP 2008 -307007, JP2008-283972, US2008-2336610, US2009-047263, j2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009- 006997, WO2009-007852) Any of the induced pluripotent stem cells known in the art can be used.

Various iPSC lines established by NIH, RIKEN, Kyoto University, Lonza, etc. are available as induced pluripotent stem cell lines. For example, human iPSC strains include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, etc., Kyoto University's 253G1 strain, 253G4 strain, 1201C1 strain, etc. 1205D1 stock, 1210B2 stock, 1383D2 stock, 1383D6 stock, 201B7 stock, 409B2 stock, 454E2 stock, 606A1 stock, 610B1 stock, 648A1 stock, 1231A3 stock, FfI-01s04 stock, QHJI01s04 stock, Lonza's TC-1133HKK_05G stock, T.C. -1133HKK_06E strain, or an iPSC strain obtained by genetically modifying the above iPSC strain.

The species of origin of pluripotent stem cells is not particularly limited, and examples include rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, dogs, and cats. The cells may be cells of felids such as humans, monkeys, rhesus monkeys, marmosets, orangutans, primates such as chimpanzees, and the like. The preferred species of origin is human.

In this specification, unless otherwise specified, "cell" includes "cell population." A cell population may be composed of one type of cell, or may be composed of two or more types of cells.

As used herein, the term "cell aggregate" refers to a structure containing three types of cells: organ cells or cells derived therefrom, vascular endothelial cells or cells derived therefrom, and mesenchymal stem cells or cells derived therefrom. means. Inside such a structure, different cells have portions where they are adjacent to each other. In the production method of the present invention, a cell aggregate can be formed by filling the above-mentioned microfiber with a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells and leaving it to stand. .

In the production method of the present invention, the cell density within the microfiber during cell filling can be appropriately set based on the ^size of the desired cell aggregate, etc. 10 ⁸ cells/mL, preferably 1×10 ⁷ cells/mL to 1×10 ⁸ cells/mL.

In the production method of the present invention, the proportion of mesenchymal stem cells in the cell population at the time of cell filling can be appropriately set based on the size and properties of the desired cell aggregate, but for example, it is between 1% and 20%. It is preferably 4% or more and 15% or less.

In addition, in the production method of the present invention, the proportion of vascular endothelial cells in the cell population at the time of cell filling can be appropriately set based on the size and properties of the desired cell aggregate, but for example, 10% or more and 60% or less. and preferably 14% or more and 48% or less.

In addition, in the production method of the present invention, the proportion of organ cells in the cell population at the time of cell filling can be appropriately set based on the size and properties of the desired cell aggregate, but for example, it is 30% or more and 90% or less. Yes, preferably 45% or more and 77% or less.

As used herein, "organoid" (also referred to as "organ bud") refers to a cell aggregate that has a new function that is not possessed by the starting cell alone used in the production method of the present invention. . Preferably, the structure is capable of differentiating into an organ upon maturation, and such differentiation ability can be determined, for example, by transplanting the structure into a living body and examining whether it can differentiate into a target organ (target organ). It can be confirmed that the organoid is differentiated into the following.

The organoid may be, for example, an organoid that differentiates into organs such as the kidney, liver, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, and spinal cord, and specifically, For example, lung organoids, liver organoids, airway epithelial organoids, intestinal organoids, pancreatic organoids, kidney organoids, brain organoids, airway organoids, gastric organoids, thyroid organoids, thymus organoids, testicular organoids, esophageal organoids, skin organoids, neural organoids, fallopian tubes. Examples include organoids, ovarian organoids, salivary gland organoids, eye vesicle organoids, optic cup organoids, bladder organoids, prostate organoids, cartilage organoids, heart organoids, bone tissue organoids, muscle tissue organoids, cancer organoids, and the like. Whether a certain structure is an organoid that differentiates into an organ can be confirmed by examining the expression of marker proteins. For example, in liver organoids, markers include HHEX, SOX2, HNF4A, AFP, ALB, etc.; in pancreatic organoids, markers include PDX1, SOX17, and SOX9; and in organoids that differentiate into the intestinal tract, markers include CDX2, SOX9, etc. In kidney organoids, Pax2, Six2, etc. become markers. Among the terms used among those skilled in the art: liver bud, liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, intestinal organoid (K. Matsumoto, et al Science.19;294(5542):559-63.(2001)) and the like are included in the organoids in the present invention.

Regarding the production of organoids as described above, for example, liver organoids can be produced based on known methods (eg, WO2013/047639, WO2019/087988). Specifically, for example, liver progenitor cells (organ cells), mesenchymal stem cells, and vascular endothelial cells are induced from pluripotent stem cells, and a mixture of these is filled into microfibers and cultured in suspension. Liver organoids can be generated.

For example, pancreatic organoids can be produced based on known methods (eg, WO2013/047639, WO2015/178431, WO2017/047797, etc.). Specifically, for example, pancreatic β cells (organ cells), vascular endothelial cells, and mesenchymal stem cells are derived from pluripotent stem cells, and a mixture of these is filled into microfibers and cultured in suspension. Pancreatic organoids can be produced.

For example, for kidney organoids, known methods (e.g. WO2019/230737, WO2020/022261, Taguchi & Nishinakamura, 2017, Cell Stem Cell 21, 730-746 December 7, 2017, Tsujimoto et al., 2020, Cell Reports 31, 107476 April 7, 2020, etc.). Specifically, for example, collecting duct progenitor cells (organ cells), nephron progenitor cells (organ cells), mesenchymal stem cells, and vascular endothelial cells are induced from pluripotent stem cells, and a mixture of these is inserted into microfibers. Kidney organoids can be produced by filling the cells into cells and culturing them in suspension.

Organoids with even more complex structures can be induced by using the above-mentioned organoids alone or in combination. Furthermore, in the production method of the present invention, cell aggregates formed within microfibers can be recovered by dissolving the microfibers with a chelating agent such as EDTA.

In the production method of the present invention, the temperature during culturing is not particularly limited as long as the desired cell aggregate is obtained, but may be, for example, 30°C to 40°C, preferably 37°C.

In the production method of the present invention, the culture period is not particularly limited as long as the desired cell aggregate is obtained, but may be, for example, 1 to 14 days, preferably 3 to 7 days.

Organoids produced by the production method of the present invention may be transplanted into non-human animals and allowed to mature within the non-human animals to produce tissues or organs. Non-human animals that can be used include mice, rabbits, pigs, dogs, monkeys, and the like. Furthermore, the non-human animal used for the maturation is preferably an immunodeficient animal in order to avoid immune rejection.

2. Transplant therapy agent Cell aggregates obtained by the production method of the present invention (hereinafter sometimes referred to as "cell aggregates of the present invention") typically contain the starting cells used in the production method of the present invention. It is a structure that can be called an organoid and has new functions (for example, the ability to secrete albumin) that have not been previously used, and can become tissues or organs by maturing in vivo. Therefore, the cell aggregate of the present invention can be suitably used for cell transplantation therapy. Therefore, in another aspect of the present invention, a cell transplant therapy agent (hereinafter sometimes referred to as "cell transplant therapy agent of the present invention") containing the cell aggregate of the present invention is provided. Furthermore, an effective amount of the cell aggregate of the present invention can be administered to or transplanted into mammals to be treated (e.g., humans, mice, rats, monkeys, cows, horses, pigs, dogs, etc.). Also encompassed by the invention are methods of treating injuries (including defects) or diseases. Furthermore, "treatment of tissue or organ damage" also includes regeneration of damaged tissue or organs.

When a cell aggregate is transplanted into a tissue or organ, the transplant site may be any site as long as it can be transplanted, such as intracranial, mesenteric, liver, spleen, kidney, under the renal capsule, etc. Examples include the upper portal vein. When transplanting, a cell aggregate produced by the production method of the present invention may be transplanted, or a tissue or organ produced by maturing the cell aggregate produced by the production method of the present invention in a non-human animal may be transplanted. You may.

When using the cell aggregate of the present invention for cell transplantation therapy, it is derived from iPS cells established from somatic cells that have the same or substantially the same HLA genotype as the recipient individual, from the viewpoint that rejection will not occur. It is desirable to use cells that undergo genetic modification or iPS cells that have been genetically modified as starting cells for the production method of the present invention. Here, "substantially the same" means that the HLA genotypes match to such an extent that the immune response to the transplanted cells can be suppressed by immunosuppressants; for example, HLA-A, HLA-B and somatic cells with HLA types that match the three loci of HLA-DR or four loci including HLA-C. If sufficient cells cannot be obtained due to reasons such as age or constitution, cell aggregates collected from microfibers can be embedded in capsules such as polyethylene glycol or silicone, or porous containers to prevent rejection. It is also possible to transplant it in a state where it is avoided.

If necessary, the cell aggregate of the present invention may be mixed with a pharmaceutically acceptable carrier according to conventional methods to produce parenteral preparations such as injections, suspensions, and drips. . Accordingly, in one embodiment, there is also provided a method for producing a cell transplantation therapeutic agent that includes the step of formulating the cell aggregate of the present invention. Such a manufacturing method may include a step of preparing a cell aggregate of the present invention. Furthermore, it can also include a step of preserving the cell aggregate of the present invention.

Pharmaceutically acceptable carriers that may be included in the parenteral formulation include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Examples include aqueous solutions for injection. The cell transplant therapy agent of the present invention includes, for example, a buffer (e.g., phosphate buffer, sodium acetate buffer), analgesic agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human (serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. Furthermore, the dosage or transplant amount of the cell transplant therapy agent of the present invention and the number of times of administration or number of transplants can be appropriately determined depending on the age, body weight, symptoms, etc. of the mammal to be administered.

The cell transplantation therapeutic agent of the present invention is provided in a state of cryopreservation under conditions commonly used for cryopreservation of cells or cell aggregates, and can also be used after being thawed before use. In that case, serum or a substitute thereof, an organic solvent (eg, DMSO), etc. may be further included. In this case, the concentration of serum or its substitute may be, but is not particularly limited to, about 1 to about 30% (v/v), preferably about 5 to about 20% (v/v). The concentration of organic solvent may be, but is not limited to, 0 to about 50% (v/v), preferably about 5 to about 20% (v/v).

The present invention will be described in more detail with reference to Examples below, but the present invention is not limited to these in any way.

<Experimental method>
1. Culture method for human induced pluripotent stem cells (iPSCs) Cryopreserved human iPSCs (QHJI01s04, obtained from Kyoto University) were immersed in hot water at 37°C for 2 minutes and thawed while shaking by hand. The cell preservation solution was suspended in StemFit AK03 medium (Ajinomoto Healthy Supply) in a volume 9 times that of the cell preservation solution, and centrifuged at 150-200×g for 5 minutes. The cell supernatant was removed, and the cells were suspended in a medium containing 10 μM Y-27632 and 0.22 to 0.25 g/cm ² iMatrix-511MG (Nippi) in AK03 medium, and seeded in a 90 mm dish. The medium was replaced with AK03 medium on the first day of culture, and thereafter, the medium was replaced on days 3, 5, and 6 of culture. Regarding passage, human iPSCs on day 7 of culture were washed with PBS, and 2 ml of Accutase (Innovative Cell Technologies) was added and treated at 37°C for 5 to 10 minutes to detach the cells. 2 ml of AK03 medium was added, the cells were transferred to a 15 ml tube, and centrifuged at 150-200 xg for 5 minutes. After removing the cell supernatant, the cells were suspended in AK03 medium supplemented with 10 μM Y-27632 and 0.22-0.25 μg/cm ² iMatrix-511MG, seeded in a 90 mm dish, and treated in the same way as after the first day of culture. Culture was carried out until the 7th day of culture.

2. Method for inducing differentiation of iPSCs into liver progenitor cells After washing human iPSCs cultured for 7 days with PBS, 2 ml of Accutase was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, remove the cell supernatant, suspend the required amount of cells in 600-1000 μl of AK03N medium, and add 100 units/ml penicillin, 100 μg/ml streptomycin, 20% StemFit For Differentiation (Ajinomoto Healthy Supply) to RPMI medium. , 2 μM CHIR99021, 33 μg/ml Activin A, 10 μM Y-27632, 0.22-0.25 μg/cm ² iMatrix-511MG was added directly to a 90 mm dish coated from the previous day. On the first day of culture, the RPMI medium was replaced with a medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 20% StemFit For Differentiation, 2 μM CHIR99021, 33 μg/ml Activin A, and 500 μM Sodium butyrate. On the third day of culture, the RPMI medium was replaced with a medium containing 100 units/ml penicillin/100 μg/ml streptomycin, 20% StemFit For Differentiation, 33 μg/ml Activin A, and 500 μM Sodium butyrate. On the fourth day of culture, the RPMI medium was replaced with a medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 20% StemFit For Differentiation, and 33 μg/ml Activin A. On the 5th day of culture, add 100 units/ml penicillin, 100 μg/ml streptomycin, 1× NEAA (MEM Non-Essential Amino Acids Solution), 1% dimethyl sulfoxide, 1 mM L-Glutamine, 100 μM to StemFit Basic03 (Ajinomoto Healthy Supply). The medium was replaced with a medium containing 2-mercaptoethanol, and the medium was changed every day until the 9th day of culture. Cells on day 10 of culture were designated as liver progenitor cells (HE).

3． Method for inducing differentiation of iPSCs into vascular endothelial cells After washing human iPSCs cultured for 7 days with PBS, 2 ml of Accutase was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, the cell supernatant was removed, and the cells were suspended in a medium containing 10 μM Y-27632 and 0.22 to 0.25 μg/cm ² iMatrix-511MG in AK03 medium, and seeded in a 90 mm dish. On the first day of culture, add 100 units/ml penicillin, 100 μg/ml streptomycin, 20% Stemfit For Differentiation, 1× GlutaMAX (ThermoFisher Scientific), 8 μM CHIR99021, and 20 ng/ml BMP-4 to DMEM/F12 medium. The medium was replaced on the third day of culture. On the 4th day of culture, the medium was changed to StemPro-34 medium (ThermoFisher Scientific) containing 100 units/ml penicillin, 100 μg/ml streptomycin, 200 ng/ml VEGF, and 2 μM Forskolin, and the medium was changed every day until the 9th day of culture. went. After washing the cells on the 10th day of culture with PBS, 2 ml of 0.05% Trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, remove the cell supernatant and transfer the cells to StemPro-34 medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 50 ng/ml VEGF, and 0.22-0.25 μg/cm ² iMatrix-511MG. Suspended and seeded in 90 mm dishes. On the 11th day of culture, the medium was replaced with MiraCell EC Culture medium (Takara Bio), and the medium was replaced every day until the 16th day of culture. Cells on day 17 of culture were designated as vascular endothelial cells (EC).

Four. Method for inducing differentiation from iPSCs to mesenchymal stem cells After washing human iPSCs cultured for 7 days with PBS, 2 ml of Accutase was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, the cell supernatant was removed, and the cells were suspended in a medium containing 10 μM Y-27632 and 0.22 to 0.25 μg/cm ² iMatrix-511MG in AK03 medium, and seeded in a 90 mm dish. On the first day of culture, 100 units/ml penicillin, 100 μg/ml streptomycin, 20% Stemfit For Differentiation, 1× GlutaMAX-I (ThermoFisher Scientific), 8 μM CHIR99021, and 50 ng/ml BMP-4 were added to DMEM/F12 medium. The medium was replaced with a new one on the third day of culture. On the fourth day of culture, add 100 units/ml penicillin, 100 μg/ml streptomycin, 20% Stemfit For Differentiation, 1× GlutaMAX, 10 ng/ml PDGF-BB, and 0.66 ng/ml Activin A to DMEM/F12 medium. The medium was replaced every day until the 5th day of culture. After washing the cells on day 6 of culture with PBS, 2 ml of 0.05% Trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, remove the cell supernatant and add 100 units/ml penicillin, 100 μg/ml streptomycin, 20% Stemfit For Differentiation, 1× GlutaMAX, 10 ng/ml PDGF-BB, 0.66 ng/ml Activin to DMEM/F12 medium. The cells were suspended in a medium supplemented with A and 10 μM Y-27632 and seeded on a 90 mm dish previously coated with 0.1% gelatin. On day 7 of culture, change the medium to DMEM/F12 medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 20% Stemfit For Differentiation, 1× GlutaMAX, 10 ng/ml bFGF, and 12 ng/ml BMP-4. The medium was replaced every day until the 9th day of culture. Cells on the 10th day of culture were used as mesenchymal stem cells (MSCs). After washing the MSCs with PBS, 2 ml of 0.05% Trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, the cell supernatant was removed, resuspended in STEM-CELLBANKER (Nippon Zenyaku Kogyo), and stored frozen at -80°C.

Five. Fluorescent staining of cells To observe cell distribution as described below, ECs were fluorescently stained with PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich), and MSCs were fluorescently stained with Vybrant DiD cell-labeling solution (Thermo Fisher Scientific). After washing ECs on day 10 of culture with PBS, 2 ml of 0.05% Trypsin-EDTA was added and treated at 37°C for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, the cell supernatant was removed, and the cells were suspended in a DMEM medium supplemented with 10% FBS. The collected ECs were fluorescently stained using the Standard Protocol of PKH67 Kit (staining reaction time: 5 minutes), and the labeled ECs were resuspended in DMEM medium supplemented with 10% FBS, and the number of cells was counted. .

Cryopreserved MSCs were immersed in 37°C hot water for 3 minutes and thawed while shaking by hand. The cell preservation solution was suspended in a DMEM medium supplemented with 10% FBS, and centrifuged at 150-200×g for 5 minutes. The collected MSCs were fluorescently stained using the Standard Protocol of Vybrant DiD cell-labeling solution (staining reaction time: 20 minutes), and the labeled MSCs were resuspended in DMEM medium supplemented with 10% FBS. The number was measured. The stained ECs and MSCs were used for the production of cell-filled hollow microfibers and spheroid production using Elplasia plates, which will be described later.

6. Collection of HE After washing HE on day 10 of culture with PBS, 2 ml of 0.05% Trypsin-EDTA was added and treated at 37°C for 3 to 10 minutes to detach the cells. After cell collection and centrifugation, the cell supernatant was removed, and the cells were resuspended in a DMEM medium supplemented with 10% FBS, and the number of cells was counted. The recovered HE was used for the production of cell-filled fibers and the production of spheroids using Elplasia plates, which will be described later.

7. Preparation of cell-filled hollow microfibers A medium containing KBM VEC-1 Basal Medium (KOHJIN BIO) supplemented with KBM VEC-1 Supplement (KOHJIN BIO) and DMEM medium at a ratio of 1:1, 2.5% FBS, 50 nM dexamethasone , 10 ng/ml Oncostatin M, and 10 μM Y-27632 were added to prepare a medium (LB medium). HE:PKH67-labeled EC:DiD-labeled MSCs were suspended in LB medium with or without 0.3% (w/v) methylcellulose at a ratio of 10:7:1, 1×10 ⁷ cells/ml, 5× Hollow microfibers with an inner diameter of 200 μm and containing a total number of cells of 5.4×10 ⁵ were produced at a cell density of 10 ⁷ cells/ml or 10×10 ⁷ cells/ml. The hollow microfiber was manufactured according to the microfiber manufacturing method described in Japanese Patent No. 5,633,077.

8. Culture of hollow microfibers filled with cells Hollow microfibers filled with 5.4×10 ⁵ cells of each cell density were placed in each well of a 24-well plate containing 0.5 ml of LB medium and cultured. On the first day of culture, 0.5 ml of LB medium was added, and from the second day to the sixth day of culture, the medium was replaced by 0.5 ml every day.

9. Preparation of spheroids (control) using Elplasia plates Suspend HE:PKH67-labeled EC:DiD-labeled MSCs in a ratio of 10:7:1 in LB medium, and place the total number of cells at 5.4x in a 24-well Elplasia plate (Corning). They were seeded at ¹⁰⁵ cells/0.5 mL/well. Cultured for 7 days. On the first day of culture, 0.5 ml of LB medium was added, and from the second day to the sixth day of culture, the medium was replaced by 0.5 ml every day.

Ten. Observation of cell morphology and distribution Observation was performed using a fluorescence microscope (Keyence) to confirm cell morphology and distribution of EC and MSC.

<Results>
The morphology and cell distribution of spheroids prepared on Elplasia plates (control) and cells filled in hollow microfibers on day 7 of culture were confirmed using a fluorescence microscope. All spheroids prepared using Elplasia plates were spherical with a size of 500 μm or less, and even in fluorescence observation, ESCs (red) and MSCs (green) were evenly distributed (Figure 1). It was confirmed that spherical spheroids with a diameter of 500 μm or less were formed within the fiber at any cell concentration in the cells filled in the hollow microfiber. In addition, fluorescence observation confirmed that EC (red) and MSC (green) were evenly distributed within each spheroid (Figure 2).

11. Examination of albumin secretion ability On the 7th day of culture, the medium of spheroids (control) prepared on Elplasia plates and cells filled in hollow microfibers was removed and replaced with fresh medium. After 24 hours, the culture supernatant was collected, and the albumin concentration in the culture supernatant was measured using a human albumin ELISA Kit (Bethyl Laboratories).

<Results>
The amount of albumin secreted per unit cell number is shown (Figure 3). Albumin secretion was confirmed under all conditions. The amount of albumin secretion per unit cell number was approximately the same as that of Control LB under medium density (5×10 ⁷ cells/ml) conditions. Furthermore, since albumin is secreted only from mature hepatocytes, it was suggested that the liver progenitor cells present in the cell aggregates matured and albumin was secreted from the cell aggregates.

According to the present invention, cell aggregates containing multiple types of cells can be efficiently produced in large quantities within hollow microfibers. Further, since the cell aggregates produced according to the present invention can function as organoids, they are extremely useful as therapeutic agents for transplantation into living bodies.

This application is based on Japanese Patent Application No. 2022-126643 (filing date: August 8, 2022) filed in Japan, the contents of which are fully included in this specification.

Claims (11)

A method for producing a cell aggregate, which includes the steps of filling a mixture of mesenchymal stem cells, vascular endothelial cells, and organ cells into microfibers and culturing them in suspension within the microfibers to form cell aggregates. The method according to claim 1, wherein the cell aggregate is an organoid. The method according to claim 1 or 2, wherein the organ cells are endodermal cells. The method according to any one of claims 1 to 3, wherein the endodermal cells are hepatocytes. The method according to any one of claims 1 to 4, wherein the microfiber has an inner diameter of 20 to 300 μm. The method according to any one of claims 1 to 5, wherein the cell density within the microfiber during cell filling is 5 x 10 ⁶ cells/mL to 4 x 10 ⁸ cells/mL. The method according to any one of claims 1 to 6, wherein the proportion of mesenchymal stem cells in the cell population at the time of cell filling is 1% or more and 20% or less. The method according to any one of claims 1 to 7, wherein the proportion of vascular endothelial cells in the cell population at the time of cell filling is 10% or more and 60% or less. The method according to any one of claims 1 to 8, wherein at least one of mesenchymal stem cells, vascular endothelial cells, and organ cells is derived from induced pluripotent stem cells. A cell aggregate produced by the method according to any one of claims 1 to 9. A transplant therapy agent comprising the cell aggregate according to claim 10.

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